Microbial Ecology (2018) 76:839–850 https://doi.org/10.1007/s00248-018-1154-8 HOST MICROBE INTERACTIONS Detecting Symbioses in Complex Communities: the Fungal Symbionts of Bark and Ambrosia Beetles Within Asian Pines James Skelton1 & Michelle A. Jusino2 & You Li1 & Craig Bateman3 & Pham Hong Thai4 & Chengxu Wu5 & Daniel L. Lindner2 & Jiri Hulcr1,3 Received: 15 August 2017 /Accepted: 31 January 2018 /Published online: 24 February 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Separating symbioses from incidental associations is a major obstacle in symbiosis research. In this survey of fungi associated with Asian bark and ambrosia beetles, we used quantitative culture and DNA barcode identification to characterize fungal communities associated with co-infesting beetle species in pines (Pinus) of China and Vietnam. To quantitatively discern likely symbioses from coincidental associations, we used multivariate analysis and multilevel pattern analysis (a type of indicator species analysis). Nearly half of the variation in fungal community composition in beetle galleries and on beetle bodies was explained by beetle species. We inferred a spectrum of ecological strategies among beetle-associated fungi: from generalist multispecies associates to highly specialized single-host symbionts that were consistently dominant within the mycangia of their hosts. Statistically significant fungal associates of ambrosia beetles were typically only found with one beetle species. In contrast, bark beetle-associated fungi were often associated with multiple beetle species. Ambrosia beetles and their galleries were frequently colonized by low-prevalence ambrosia fungi, suggesting that facultative ambrosial associations are commonplace, and ecological mechanisms such as specialization and competition may be important in these dynamic associations. The approach used here could effectively delimit symbiotic interactions in any system where symbioses are obscured by frequent incidental associations. It has multiple advantages including (1) powerful statistical tests for non-random associations among potential symbionts, (2) simultaneous evaluation of multiple co-occurring host and symbiont associations, and (3) identifying symbionts that are significantly associated with multiple host species. Keywords Bark beetles . Microbiome . Mutualism . Ophiostomatales . Platypodinae . Scolytinae Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00248-018-1154-8) contains supplementary material, which is available to authorized users. Identifying symbioses under conditions of natural complexity is difficult. Simply defining the term Bsymbiosis^ has been a * Jiri Hulcr contentious topic since its inception, though the current [email protected] trend—and our preference—is for a broad definition such as De Bary’s original usage, which includes interactions with 1 School of Forest Resources and Conservation, University of Florida, positive as well negative outcomes [1–3]. Regardless of the Gainesville, FL 32603, USA precise definition used, the first condition necessary for sym- 2 United States Forest Service, Northern Research Station, Center for biosis is that the organisms occur in proximity in nature. Forest Mycology Research, One Gifford Pinchot Drive, Madison, WI 53726, USA Surveys of microbial symbionts often verify this first condi- tion alone. Here, we go one step further and employ empirical 3 Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA tests for significant statistical relationships among hosts and potential symbionts. We posit that species that are co-adapted 4 Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Cau Giay, Hanoi, Vietnam and/or co-dependent upon one another will be found together more often than expected by chance, given their respective 5 Key Laboratory of Forest Protection of State Forest Administration, Research Institute of Forest Ecology, Environment and Protection, frequencies among samples. The null hypothesis of chance Chinese Academy of Forestry, Beijing, China associations for observed associations can be tested with 840 Skelton J. et al. freely available permutation-based statistical software and stage, management regime, land cover, or experimental treat- quantitative survey data. We demonstrate the value of this ments, but any grouping scheme can be used to identify species approach in a survey of the fungi associated with co- with non-random associations to factors of interest [35, 36]. We infesting bark and ambrosia beetles of recently dead pines used MPA to test for non-random associations among host (Pinus) in Asian forests, though the same approach could be beetle species and potential fungal symbionts, while simulta- applied to any system where microbial symbioses are diverse, neously evaluating multiple associations to characterize the variable in the breadth of species involved, and hidden among breadth of associations among multiple beetle and fungal taxa. frequent incidental associations. Bark and ambrosia beetles, which comprise all ~ 7400 spe- cies of the Curculionidae subfamilies Scolytinae and Methods Platypodinae [4], have associations with fungi that range from incidental commensalism, to parasitism, to co-evolved obligate Study Sites and Collection Methods nutritional mutualisms [4–7]. Like many symbioses, interac- tions between beetles and fungi may change with ecological We conducted replicated quantitative culture-based sampling of context [8–13]. The term Bambrosia beetle^ refers to scolytine beetle bodies and galleries to test for significant associations and platypodine beetles that farm gardens of Bambrosia^ fungi between beetle species and fungi in Asian pine forests in Tam within the galleries they excavate through the nutrient-poor Dao National Park, Hồ Sơn, Vinh Phuc Province, Vietnam, and xylem or pith of their plant hosts [14]. Ambrosia beetles do Fuzhou, Fujian Province; Guiyang and Weining, Guizhou not eat wood, but instead eat fungi either exclusively or nearly Province; and Nanshan, Shaanxi Province, China. During two exclusively. Reciprocally, ambrosia fungi depend on beetles for collecting trips in March 2016 in Vietnam, we collected bark dispersal and colonization of new plant tissues [15–17]. The and ambrosia beetles and their galleries from the trunks of eight fungal mutualists are transported to new gardens inside pouch- dead or moribund pines within 1 km2 in Tam Dao National or pit-like organs, termed mycangia, which support growing Park. Plantations of Chinese red pine (Pinus massoniana) ambrosia fungi [17]. The ambrosia habit has evolved indepen- formed most of the forest canopy at this site. During collections dently in at least 12 lineages of scolytine and platypodine bee- from August 2015 to December 2015 in Central and Southern tles [18, 19] and in at least 9 lineages of fungi [20–24], and China, Xyleborus pinicola and Orthotomicus chaokhao were those estimates are likely to increase as more taxa are studied collected from Chinese red pine in Gushan Mt., Fuzhou, Fujian [4]. Many ambrosia beetle species appear to be obligately as- Province; X. pinicola and Euwallacea fornicatus were collected sociated with one or a few fungal taxa [21, 24], though they are from Chinese red pine in Huaxi, Guiyang, Guizhou Province; often associated with additional commensals [25, 26], parasites Polygraphus spp., Tomicus yunnanensis, Tomicus minor,and [27], and facultative mutualists [8, 15, 28, 29]. Hylurgops longipillus were collected from Yunnan pine (Pinus In contrast to ambrosia beetles which bore into xylem, bark yunnanensis) in Weining, Bijie, Guizhou Province; and beetles consume relatively nutrient-rich phloem and typically Dendroctonus armandi was collected from Chinese white pine do not depend exclusively on fungi for nutrition [5]. However, (Pinus armandii) in Huoditang, Nanshan, Shaanxi Province. some bark beetle species benefit from mutualistic nutritional Bark-dwelling beetles were located by removal of sections fungi as a key component of their diet [30–33], while other of bark. Xylem-inhabiting ambrosia beetles and their galleries associated fungi may be parasites or competitors of bark bee- were excised from the trees using chisels. Individual beetles tles and their mutualists [34]. Thus, the relationships between and sections of their galleries were placed aseptically into bark beetles and fungi are often more variable and dependent sterile 1.5-ml centrifuge tubes modified with small holes on context. Fungal mutualisms as well as parasitic associa- through the cap to permit gaseous exchange. To maintain sta- tions may be more prevalent among bark beetles than we tistical independence among replicated observations, only one currently know, but they are difficult to identify because of representative beetle and gallery sample were taken from ex- inherent variability and the ubiquitous presence of incidental tensive galleries with multiple beetles, and galleries were sam- environmental taxa. Consequently, improved observational pled from multiple trees for each beetle species. Beetles methods, replicated sampling, and rigorous tests of appropri- remained in their galleries and were stored alive, in the dark, ate null hypotheses are needed to tease apart these complexes at ambient temperature until processing, which was no longer and often obfuscated relationships. than 6 days after